Characterization of a cell-surface protein antigen of hydrophilic Streptococcus mutans strain GS-5

Fourteen strains of Streptococcus mutans serotype c were examined for their cell-surface protein antigens in terms of hydrophobicity, Mr and immunochemical specificities. Thirteen strains were hydrophobic, while strain GS-5 was markedly hydrophilic as compared to the other strains tested. Cell-surface protein antigens were then analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting. A protein antigen of Mr 190,000 (PAc) was found in cell extracts and culture supernatants of all the hydrophobic strains. Neither culture supernatant nor cell extract of strain GS-5 contained PAc. Strain GS-5, however, produced extracellularly a large amount of a protein of Mr 155,000 (PAGS-5) which reacted with rabbit anti-PAc serum. Immunodiffusion analysis showed that PAGS-5 lacked a part of the antigenic moieties in the PAc molecule. SDS-PAGE and radioimmunoassay showed a small amount of PAGS-5 on the cell surface of strain GS-5. These findings suggest that PAGS-5 may correspond to PAc which lacks a region participating in binding of PAc to the streptococcal cell.     

Insight into heterogeneity in cell-surface hydrophobicity and ability to degrade hydrocarbons among cells of two hydrocarbon-degrading bacterial populations

The sequential bacterial adherence to hydrocarbons (BATH) of successive generations of hydrophobic fractions of Paenibacillus sp. R0032A and Burkholderia cepacia gave rise to bacterial populations of increasing cell-surface hydrophobicity. Thus, hydrophobicity of the first generation (H1) was less than that of the second generation (H2), which was less than that of the third generation (H3). Beyond H3, the hydrophobic populations became less stable and tended to lyse in hexadecane after violent (vortex) agitation, resulting in an apparent decline in BATH value. The exhaustively fractionated aqueous-phase population (L) was very hydrophilic. The overall cell-surface distribution of the population was L < parental strain < H1 < H2 < H3. The ability to degrade crude oil, hexadecane, or phenanthrene matched the degree of cell-surface hydrophobicity: L < P < H1 < H2 < H3. Thus, in natural populations of hydrocarbon-degrading Paenibacillus sp. R0032A and B. cepacia, there is a heterogeneity in the hydrophobic surface characteriistics that affects the ability of cells to use various hydrocarbon substrates.     

Sequential hydrophobic partitioning of cells of Pseudomonas aeruginosa gives rise to variants of increasing cell-surface hydrophobicity

The partitioning of bacterial cells in a dual aqueous-solvent phase system leads to separation into 'hydrophilic' and hydrophobic functions. Sequential multistep partitioning, accompanied by successive enrichment, gives rise to several cycles of hydrophobic and hydrophilic cell populations which possess different cell-surface hydrophobicity characteristics. Characterization of the cell-surface hydrophobicity by several methods (salting-out aggregation test, bacterial adherence to hydrocarbon, polystyrene binding and hydrophobic interaction chromatography) was carried out. The cell-surface hydrophobicity varied in the order: hydrophilic fraction < parental strain < first cycle hydrophobic variant < second cycle hydrophobic variant < third cycle hydrophobic variant. Electron microscopy showed that the most hydrophobic variant was densely covered by hydrophobic structures - fimbriae - whereas the parental strain was covered by a mixture of surface structures. The hydrophilic variant was covered by an amorphous exopolymeric substance, which is a polysaccharide, shown by its reaction with Alcian blue.     

Surface properties of Staphylococcus aureus affecting chemiluminescence response of human phagocytes

To assess the surface properties of Staphylococcus aureus affecting the response of human phagocytes, the effects of the organisms with different surface properties on the chemiluminescence (CL) response of human phagocytes were examined. The magnitude of the phagocytic CL response to hydrophobic strains was significantly greater than that to hydrophilic strains, while no significant difference in the CL response was seen between protein A-deficient strains and their parent strains. The CL response to the hydrophilic organisms prepared from a hydrophobic strain by trypsin treatment decreased significantly. These results suggest that the phagocytic CL response to staphylococci depends on the hydrophobicity of the surface, but not on the presence of protein A. Two protein A-deficient strains which were isolated from protein A-positive strains showed identical hydrophobicity with their parent strains. All of the hydrophilic strains isolated from hydrophobic strains possessed protein A identical to that of their parent strains. Moreover, a hydrophilic strain could be isolated from a protein A-deficient, hydrophobic strain. These results strongly suggest that protein A is not solely responsible for the surface hydrophobicity of S. aureus.     

Contribution of cell surface protein antigen PAc of Streptococcus mutans to bacteremia

Streptococcus mutans, a major cariogenic bacterium, is occasionally isolated from the blood of patients with bacteremia and infective endocarditis. Mutant strains of S. mutans MT8148, defective in the major surface proteins glucosyltransferase (GTF) B-, C-, and D-, and protein antigen c (PAc), were constructed by insertional inactivation of each respective gene with an antibiotic resistant cassette. Susceptibility to phagocytosis was determined by analyses of interactions of the bacteria with human polymorphonuclear leukocytes, and the PAc-defective mutant strain (PD) showed the lowest rate of phagocytosis. Further, when PD and MT8148 were separately injected into the jugular veins of Sprague-Dawley rats, PD was recovered in significantly larger numbers and for a longer duration, and caused more severe systemic inflammation than MT8148, indicating that S. mutans PAc is associated with its systemic virulence in blood. Next, 100 S. mutans clinical isolates from 100 Japanese children and adolescents were analyzed by Western blotting using antisera raised against recombinant PAc, generated based on the pac sequence of MT8148. Four of the 100 strains showed no positive band and each exhibited a significantly lower phagocytosis rate than that of 25 randomly selected clinical strains (P < 0.01). In addition, three of the 100 strains possessed a lower molecular weight PAc and a significantly lower rate of phagocytosis than the 25 reference strains (P < 0.05). These results suggest that S. mutans PAc may be associated with phagocytosis susceptibility to human polymorphonuclear leukocytes, with approximately 7% of S. mutans clinical isolates possible high-risk strains for the development of bacteremia.     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37 degrees C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.     

Molecular characterization of a surface protein antigen gene from serotype c Streptococcus mutans, implicated in dental caries

The complete nucleotide sequence of the gene for a cell-surface protein antigen (PAc) of Streptococcus mutans MT8148 (serotype c) was determined. The pac gene consisted of 4695 bp and coded for a 170773D protein. The pac gene product contained a putative 38 amino acid signal peptide, resulting in a 166817D mature protein. A potential promoter sequence and a putative Shine-Dalgarno sequence preceded the open reading frame. Two internal repeating amino acid sequences were present in the PAc. One repeating region located in the N-terminal region was rich in alanine, and the other located in the central region was rich in proline. Southern blot analysis under the less stringent condition (allowing up to 35% base mismatch) revealed that the probe covering the proline-rich region hybridized to DNA preparations from strains of Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei as well as Streptococcus mutans.     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37°C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.     

Role and behaviour of the hydrophobic conditions in bacterial adhesion to incurrent siphon in a bivalve mollusc

Surface hydrophobicity is a widely distributed characteristic among human bacterial pathogens playing an important role in microbes retention by the incurrent siphon of a marine bivalve. Feeding retention experiments with the bivalve Mesodesma donacium was done with hydrophobic strains alone (Staphylococcus aureus, Salmonella paratyphi, Vibrio cholerae) or with mixed cultures using a hydrophilic strain (Aeromonas hydrophila). Results showed that hydrophobic bacteria are retained in greater numbers than hydrophilic bacteria. This difference is statistically significant. Hydrophobic strains also survive longer than hydrophilic ones in sea water. Surface hydrophobicity is to be considered as a factor influencing concentration of hydrophobic pathogens by filter feeding molluscs.     

Autoaggregation,hydrophobic, and hydrophylic properties of Moraxella catarrhalis strains

The hydrophobicity of the bacterial cell surface was evaluated via the salt aggregation test (SAT) in 58 strains (19 from the lower and 39 from the upper respiratory tracts) of Moraxella catarrhalis in hospitalized patients aged 25 to 65. Based on the SAT results, the strains were divided into three groups: autoaggregating (highly hydrophobic), hydrophobic, and hydrophilic. At a temperature of 37 degrees C, the autoaggregating, hydrophobic or hydrophilic properties did not depend on the choice of a medium, whereas at 22 degrees C the investigated properties did (p<0.0001). Taking into account the origin of the strains (lower vs. upper respiratory tract), it was found that: in the strains cultivated in liquid medium, both highly hydrophobic, hydrophobic and hydrophilic surfaces were present with a comparative frequency, independent of the strain isolation site and cultivation conditions; strains with highly hydrophobic and hydrophobic surfaces, but only those cultivated on solid media at 22 degrees C, were much more often isolated from sputum rather than from nose and throat swabs, whereas a statistically significant incidence of hydrophilic strains was found in samples from the upper rather than lower respiratory tract.     

Surface properties of lactobacilli isolated from the small intestine of pigs

One hundred wild-type strains of the genus Lactobacillus were isolated from the small intestine of newly-slaughtered pigs up to 6 months of age. Cell surface hydrophobicity and capsule formation were studied on a number of strains. Strains showing high surface hydrophobicity as measured by the salt-aggregation test and hydrophobic interaction chromatography on Octyl Sepharose were commonly found to adhere in high numbers to isolated pig intestinal epithelial cells. Heat and protease treatment of bacteria of high surface hydrophobicity, including autoaggregating strains in phosphate-buffered saline, showed a drastic decline in this surface property. Three hydrophilic strains (LBp 1044, 1068 and 1073) also showed binding to intestinal cells but at a lower level (approx. 5 bacteria/cell) as compared with the best binding hydrophobic strain (LBp 1063, approx. 11 bacteria/cell). These findings suggest that different or multiple adhesion mechanisms may be involved in the colonization of the small intestinal mucosa of pigs. Cultures of selected strains grown in liquid media rich in carbohydrates did not affect their hydrophobic cell surface character. Therefore it seems less likely that carbohydrate capsule polymers are the major determinants of intestinal colonization of lactobacilli in pigs.     

Surface properties of lactobacilli isolated from the small intestine of pigs

One hundred wild-type strains of the genus Lactobacillus were isolated from the small intestine of newly-slaughtered pigs up to 6 months of age. Cell surface hydrophobicity and capsule formation were studied on a number of strains. Strains showing high surface hydrophobicity as measured by the salt-aggregation test and hydrophobic interaction chromatography on Octyl Sepharose were commonly found to adhere in high numbers to isolated pig intestinal epithelial cells. Heat and protease treatment of bacteria of high surface hydrophobicity, including autoaggregating strains in phosphate-buffered saline, showed a drastic decline in this surface property. Three hydrophilic strains (LBp 1044, 1068 and 1073) also showed binding to intestinal cells but at a lower level (approx. 5 bacteria/cell) as compared with the best binding hydrophobic strain (LBp 1063, approx. 11 bacteria/cell). These findings suggest that different or multiple adhesion mechanisms may be involved in the colonization of the small intestinal mucosa of pigs. Cultures of selected strains grown in liquid media rich in carbohydrates did not affect their hydrophobic cell surface character. Therefore it seems less likely that carbohydrate capsule polymers are the major determinants of intestinal colonization of lactobacilli in pigs.     

Role of hydrophobic surface proteins in mediating adherence of group B streptococci to epithelial cells.

Determination of the cell-surface hydrophobicity of group B streptococci by hydrophobic interaction chromatography on phenyl-Sepharose revealed that human and bovine group B streptococcal isolates with protein surface antigens, either alone or in combination with polysaccharide antigens, were mainly hydrophobic, whereas those with polysaccharide antigens alone were mainly hydrophilic. Removal of capsular neuraminic acid enhanced, and pronase treatment reduced, surface hydrophobicity. The hydrophobic surface proteins, solubilized by mutanolysin treatment of the bacteria and isolated by hydrophobic interaction chromatography, appeared in SDS-PAGE as numerous protein bands. Staphylococcal carrier cells loaded with antibodies produced against hydrophobic surface proteins agglutinated specifically with hydrophobic group B streptococci. No agglutination reaction was observed with hydrophilic cultures. Hydrophobic group B streptococci adhered to buccal epithelial cells in significantly higher numbers than did hydrophilic cultures. The adherence of group B streptococci to epithelial cells was inhibited in the presence of isolated hydrophobic proteins and in the presence of specific antibodies produced against hydrophobic proteins. The results of this study demonstrate a close relation between the occurrence of type-specific antigens, surface hydrophobicity and the adherence of group B streptococci to epithelial cells.     

Compositional factors influencing cell surface hydrophobicity ofPasteurella multocida variants

The influence of macromolecules other than lipopolysaccharide on the hydrophobic properties ofPasteurella multocida was investigated by assessing cell surface hydrophobicity (CSH) after experimentally modifying surfaces of various strains. CSH of hydrophobic variants was enhanced by growth on blood-supplemented medium and mechanical shearing, whereas chloramphenicol, oxytetracycline, trypsin, and pronase E treatments decreased CSH. No such modifications were observed for hydrophilic strains. Microscopic observations revealed hydrophilic strains to be heavily encapsulated in contrast to hydrophobic strains. Repeated subculturing reduced encapsulation with a concomitant increase in CSH for one hydrophilic strain while exerting no changes in the other hydrophilic strain examined. Hyaluronidase removal of capsular material from a serotype A strain resulted in increased CSH; subsequent exposure to pronase E resulted in partial restoration of hydrophilicity. These data suggest the encapsulation of hydrophilicP. multocida strains masks a relatively hydrophobic surface that is conferred, at least in part, by the presence of one or more surface-exposed proteins common to both hydrophilic and hydrophobic variants.     

Cell surface components of Streptococcus sanguis: relationship to aggregation, adherence, and hydrophobicity.

Cell surfaces of aggregation, adherence, and hydrophilic variants of Streptococcus sanguis were compared with cell surfaces of the parent strain with regard to their protein and antigenic constituents. Cell surface molecules were released by digestion with mutanolysin. Extraction with sodium dodecyl sulfate (SDS) urea, lithium diiodosalicylate, and boiling water did not solubilize any material which stained with AgNO3 in an SDS-polyacrylamide gel electrophoresis gel. The parent organism S. sanguis 12, which aggregates in saliva, adheres to saliva-coated hydroxyapatite and is hydrophobic, was found to possess a prominently staining 160,000 molecular weight (MW) protein. This protein was almost completely absent from strain 12na, a hydrophobic nonaggregating variant, and was completely absent from the hydrophilic nonaggregating strain 12L. Trypsinization of strain 12 resulted in the coincident loss of the 160,000-MW protein and the ability to aggregate in saliva. Trypsin treatment reduced but did not eliminate the hydrophobic character of the cells. Boiling destroyed their ability to aggregate, but did not alter their hydrophobicity. Cell wall digests of strain 12 contained a number of proteins which were absent from strains 12na and 12L. Mutanolysin digests of cell walls of the hydrophilic strains contained almost no material that was visible in a silver-stained SDS-polyacrylamide gel electrophoresis gel. Culture supernatants contained a number of proteins which were immunologically cross-reactive with cell surface proteins. The hydrophilic organisms released a number of 60,000- to 90,000-MW proteins not seen in culture supernatants from the parent strain.     

Starvation-Induced Effects on Bacterial Surface Characteristics

Changes in bacterial surface hydrophobicity, charge, and degree of irreversible binding to glass surfaces of seven marine isolates were followed during starvation. The degree of hydrophobicity was measured by hydrophobic interaction chromatography and by two-phase separation in a hexadecane-water system, whereas changes in charge were measured by electrostatic interaction chromatography. All isolates underwent the starvation-induced responses of fragmentation, which is defined as division without growth, and continuous size reduction, which results in populations with increased numbers of smaller cells. The latter process was also responsible for a significant proportion of the total drop in cell volume; this was observed by noting the biovolume (the average cell multiplied by the number of bacteria) of a population after various times of starvation. Four strains exhibited increases in both hydrophobicity and irreversible binding, initiated after different starvation times. The most hydrophilic and most hydrophobic isolates both showed a small increase in the degree of irreversible binding after only 5 h, followed by a small decrease after 22 h. Their hydrophobicity remained constant, however, throughout the entire starvation period. On the other hand, one strain, EF190, increased its hydrophobicity after 5 h of starvation, although the degree of irreversible binding remained constant. Charge effects could not be generally related to the increase in irreversible binding. Scanning electron micrographs showed a large increase in surface roughness throughout the starvation period for all strains that showed marked changes in physicochemical characteristics.     

一株耐热石油烃降解菌的细胞疏水性及乳化、润湿作用研究

从胜利油田油水样中分离到一株能够在60℃高温条件下利用烃类产生生物表面活性剂的菌株芽孢杆菌(Bacillus sp.)A1.结果表明:A1的细胞表面具有很强的疏水性,这有助于菌体细胞对烃类的摄取.该菌株对石油烃具有良好的乳化作用,并可在20%的高盐环境和100℃高温条件下仍显示很高的乳化活性.同时,A1可明显改变油藏岩石表面的润湿性,使其亲水性显著增强.对油藏中的岩石模拟试片石英、灰岩和玻璃作用后的接触角均减小60%以上.油藏中岩石的润湿性能增强,水驱油时更易于剥落滞留在岩石表面上的油滴或油膜,从而提高石油采收率.     

Cell Surface Hydrophobicity as a Pellicle Formation Factor in Film Strain of Saccharomyces

The film strain of Saccharomyces grows through non-film and film stages. The differences in cell surface hydrophobicity were examined at both stages. The degree of hydrophobic was quantitatively determined by comparing distribution ratios of cells between buffered aqueous and organic solvent phases. The cell surface in the film stage was more hydrophobic than that in the non-film stage, whereas the inherent non-film strain of Saccharomyces always showed low hydrophobicity. These results indicate that the change from non-film to film stage was due to a change in cells from hydrophilic to hydrophobic. The effects of growth conditions on hydrophobicity were further examined with the film strain Saccharomyces bayanus. Ethanol as sole carbon source more efficiently increased hydrophobicity than glucose. The increase in hydrophobicity seemed to depend upon respiration accompanying assimilation of ethanol. It was also found that the addition of a limited amount of biotin, as well as higher pH in medium lowered hydrophobicity. Variation in degree of pellicle formation was positively related to that of cell surface hydrophobicity.